KR20210149752A - Systems and methods for isolating microvessels from adipose tissue - Google Patents

Abstract

Methods and systems for dissociating tissues by isolating microvessels using concentrated or purified enzymes are described. The systems and methods include a second digestion, a second centrifugation operation, and a second digestion operation to digest the upper layer resulting from the first digestion and first centrifugation operation with a concentrated or purified enzyme to produce a second fat-enzyme solution, and 2 involves separation of microvessels from pellets produced by a centrifugation operation. The systems and methods can include washing the second fat-enzyme solution with an enzyme inhibitor in a post-disintegration wash.

Description

Systems and methods for isolating microvessels from adipose tissue

This disclosure claims the benefit of U.S. Provisional Application Serial No. 62/831,765, filed April 10, 2019, entitled “System and Method for Isolating Microvessels from Adipose Tissue,” the entire contents of which are herein The specification is incorporated by reference.

The present disclosure relates to a method for isolating microvessels from adipose tissue, and more particularly, to a method for isolating microvessels from adipose tissue using a concentrated or purified enzyme.

A practical method for isolating cells including microvessels from tissue is to use crude preparations of tissue dissociation enzymes to dissociate the tissue into individual cellular components.

There is a need for alternative, high-quality, efficient and effective methods to dissociate tissues and isolate microvessels.

According to the subject matter of the present disclosure, the methods and systems use concentrated or purified enzymes to isolate microvessels to dissociate tissues. The systems and methods may include a double digestion feature and/or an immediate post-digestion wash feature.

According to one embodiment, a system for dissociating tissue by isolating microvessels using an enriched enzyme comprises one or more processors, a non-transitory memory communicatively coupled to the one or more processors, and a non-transitory memory. may include machine-readable instructions stored in Concentrated enzymes may include concentrated or purified enzymes. The machine readable instructions, when executed by the one or more processors, may cause the system to perform at least the following operations as one or more protocols, wherein at least the following operations break down the ground fat in the first digest into a concentrated enzyme to thereby cause the first fat - producing an enzyme solution, centrifuging the first fat-enzyme solution from the first digestion in a first centrifugation operation to produce one or more first pellets and a layer of fat disposed over the one or more first pellets, a second operation degrading the upper fat layer with the enzyme concentrated in the digestion to produce a second fat-enzyme solution, centrifuging the second fat-enzyme solution from the second digestion in a second centrifugation operation to produce one or more second pellets operation, and passing one or more portions of one or more first pellets and one or more second pellets through one or more screens to create a plurality of discrete microvessels.

According to another embodiment, the method of dissociating the tissue by separating microvessels using the concentrated enzyme comprises the steps of decomposing the fat pulverized in the first digestion with the concentrated enzyme to produce a first fat-enzyme solution, the first centrifuging the first fat-enzyme solution from the first digest in a centrifugation operation to produce one or more first pellets and an upper fat layer disposed over the one or more first pellets, and an upper fat layer with the enzyme concentrated in a second digest decomposing to produce a second fat-enzyme solution. The method comprises centrifuging the second fat-enzyme solution from the second digestion in a second centrifugation operation to produce at least one second pellet, and at least one first pellet and at least one portion of the at least one second pellet. The method may further comprise passing through one or more screens to create a plurality of discrete microvessels.

According to another embodiment, the method of dissociating tissue by isolating microvessels using a concentrated enzyme comprises the steps of decomposing the fat pulverized in the first digestion with the concentrated enzyme to produce a first fat-enzyme solution; 1 using an additional enzyme as a catalyst for digestion of the fat-enzyme solution, centrifuging the first fat-enzyme solution from the first digestion in a first centrifugation operation onto the at least one first pellet and the at least one first pellet creating a disposed top fat layer, centrifuging the first fat-enzyme solution from the first digest in a first centrifugation operation to produce at least one first pellet and an upper fat layer disposed over the at least one first pellet; and digesting the upper fat layer with the enzyme concentrated in the second digestion to produce a second fat-enzyme solution. The method includes washing the second fat-enzyme solution with an enzyme inhibitor in a post digestion wash, centrifuging the second fat-enzyme solution from the second digestion in a second centrifugation operation to produce one or more second pellets and passing one or more portions of the one or more first pellets and the one or more second pellets through one or more screens to create a plurality of discrete microvessels. The enzyme inhibitor of the post-cleavage wash may comprise one or more peptide inhibitors, one or more small molecule inhibitors, one or more natural matrix substance inhibitors, or a combination thereof.

These and additional features provided by embodiments of the present disclosure will be more fully understood in consideration of the following detailed description in conjunction with the drawings.

The present systems and methods utilizing concentrated or purified enzymes and dual digestion features provide significantly significant isolated microvascular composition, lot-to-lot consistency, and cost-effectiveness compared to previous methods using crude enzymes.

BRIEF DESCRIPTION OF THE DRAWINGS The following detailed description of specific embodiments of the present disclosure is best understood when read in conjunction with the following drawings, wherein like structures are denoted by like reference numerals, in which:
1 depicts a flow diagram of a method of dissociating tissue by isolating microvessels using a concentrated or purified enzyme in accordance with one or more embodiments as shown and described herein.
2 depicts a flow diagram of another method of dissociating tissue by isolating microvessels using concentrated or purified enzymes in accordance with one or more embodiments as shown and described herein. and,
3 schematically illustrates a system for implementing a computer-based process for automating the method of the flowcharts of FIGS. 1 and 2 in accordance with one or more embodiments as shown and described herein;

In accordance with the embodiments described herein, systems and methods are described for isolating intact and functional microvessels using concentrated or purified enzymes to dissociate tissues such as adipose tissue or other tissues. While such enzymes may be described herein as concentrated, purified enzymes are considered to be a type of concentrated enzyme as compared to crude enzymes that exist alone without concentration or purification, and it is within the scope of this disclosure. The systems and methods may include the use of a dual breakdown of ground fat as described in more detail below. Additionally or alternatively, the systems and methods may include the use of an immediate post-disintegration wash as described in more detail below.

In operations involving crude enzymes, such as those commercially available through WORTHINGTON BIOCHEMICAL, Lakewood, NJ, ground fat can be single-digested into crude enzymes and centrifuged to produce a bottom pellet and a disposable top layer. . The disposable top layer is discarded, and one or more portions of the bottom pellet can be passed through a screen to dissociate the tissue and isolate the microvessels. An example of a method involving a single dissociation of microvessels using coenzyme to dissociate tissue is described in Hoying JB, Boswell CA, and Williams SK's paper, the angiogenic potential of microvascular fragments established in three-dimensional collagen gels ( Angiogenic Potential of Microvessel Fragments established in Three-Dimensional Collagen Gels), In Vitro Cell Dev. Bio. Anim July 1996, 32(7): 406-19.

The systems and methods described herein relate to the double digestion feature of a method for dissociating tissue by isolating microvessels using a concentrated or purified enzyme, wherein the double digestion feature is second digestion ) and the use of the top layer resulting from the first digestion. In an embodiment, the concentrated or purified enzyme comprises a concentrated or purified collagenase. In an embodiment, the concentrated or purified enzyme comprises a chromatographically concentrated or purified collagenase. In an embodiment, the concentrated or purified enzyme comprises a low protease, concentrated collagenase product of Clostridium histolyticum useful for isolating cells from tissue. In certain embodiments, the concentrated or purified enzyme comprises Collagenase Gold or DEGold Collagenase or Collagenase HA (as commercially available through VITACYTE, Indianapolis, Indiana). In embodiments, in addition to or as an alternative to Clostridial collagenase, the concentrated or purified enzyme may include Vibrio and/or other bacterial sources such as mammalian collagenase.

Referring first to Figure 1. 1 is a flowchart of a method 100 for dissociating tissues by isolating microvessels using a concentrated or purified enzyme. In block 102, the ground fat is subjected to a first digestion with a concentrated or purified enzyme to produce a first fat-enzyme solution. In embodiments, the fat is milled, for example, via manual mixing or liposuction cannula, and broken down into concentrated or purified enzymes. Embodiments of a shredding (eg, comminution) technique for crushing adipose tissue may include the use of other mechanical techniques as would be understood by one of ordinary skill in the art. Such shredding techniques can be adapted to accommodate fat for shredding in one or more procedures, such as liposuction (eg, lipoaspiration) or abdominoplasty. In the context of liposuction, a surgical cannula may be used to collect the fat that is then crushed as a liposuction. In the context of abdominoplasty, the fat mass may be removed, separated, and then further shredded.

The first fat-enzyme solution may further comprise the use of other enzymes as contaminants to assist the concentrated or purified enzymes as catalysts for degradation. Another enzyme may be, but is not limited to, deoxyribonuclease (DNase), which is a nuclease enzyme capable of hydrolyzing phosphate diester bonds connecting nucleotides, and more specifically, phosphate diester bonds in the DNA backbone. It catalyzes the hydrolytic cleavage of DNA and degrades DNA. For digestion, a digestion flask containing a fat volume matching the volume of the concentrated or purified enzyme may be used, and a stir bar that may be coated with Teflon or steel may be used. In one embodiment, the digestion flask may or may not be shaken in a water bath at 37° C. for 8-10 minutes.

In block 104, the first fat-enzyme solution of block 102 is centrifuged in a first centrifugation operation to produce one or more pellets from the first centrifugation operation and an upper fat layer disposed over the one or more pellets. Through the first centrifugation operation, the separated material including fat and microvessels may be separated.

In block 106, the upper layer resulting from the first centrifugation operation is subjected to a second digestion with a concentrated or purified enzyme to produce a second fat-enzyme solution. In an embodiment, the upper layer is the upper adipose layer that is degraded in a second digestion with fresh concentrated or purified enzymes to completely release the microvessels from the upper adipose layer.

In block 108, the second fat-enzyme solution is centrifuged in a second centrifugation operation to produce one or more pellets from the second centrifugation operation. In one embodiment, the lipid/top layer (eg, top lipid layer) and supernatant of the second fat-enzyme solution are aspirated leaving one or more pellets at the bottom of the tube.

In block 110, a portion of the one or more pellets resulting from the first centrifugation operation and the second centrifugation operation as the resulting pellets are passed through one or more screens as isolated microvessels. In one embodiment, the resulting pellet is washed with a gelatin solution and passed through two screens to collect microvessels. In an embodiment, as described in more detail below with respect to FIG. 2 , microvascular enzymes are inhibited and/or quenched to prevent collagen degradation of microvessels by cryopreservation and/or washing after digestion with enzyme inhibitors or can be minimized Cryopreservation can preserve microvessels for up to a period such as a month.

Example 1

In an exemplary procedure, 20 milliliters (ml) of ground adipose tissue is used. A first fat-enzyme solution is prepared in block 102 using 30 ml of 30 mg gold collagenase as a concentrated or purified enzyme and 30 mg DNase in 0.1% DPBS as a balanced salt solution for processing and culturing mammalian cells. Next, add 10 ml of the first fat-enzyme solution to 10 ml of fat (eg, ground fat) in the first flask to obtain 15 mg of concentrated or purified enzyme (eg, 1.0x enzyme mix) use In the first flask, 7.5 ml of the first fat-enzyme solution is added to the other 10 ml of fat so that 7.5 mg of the concentrated or purified enzyme is used (eg 0.5x enzyme mix). In block 102, both flasks are digested in a water bath at 37° C. for 8 minutes with 1 minute shaking and 7 minutes without shaking. Both flasks still retain tissue mass.

In a next step in block 104, the material in both flasks is centrifuged in a first centrifugation operation. A 1.0x enzyme mix yields larger pellets than a 0.5x enzyme mix. Adipose tissue remains on top of each centrifugation mix. At block 106, the upper layer is transferred to a new flask, the remaining enzyme of the first fat-enzyme solution is added to 7.5 ml and re-dissolved. This re-degradation acts as a second degradation feature and produces quality tissue with most of the fat cells on top, including an 8-minute shaking. After block 104, 1.0x enzyme mix produces cell clusters without microvessels in the pellet, and 0.5x enzyme mix produces larger cell clusters without microvessels in the pellet. After block 106, after the second digestion, after centrifugation of block 108 and separation of microvessels of block 110, a significant number of microvessels are produced, many of which are long. Divide the number of 45 by 0.02 ml and multiply by 40 ml, it is estimated that there are 90,000 microvessels. The resulting microvessels are pelleted, frozen in 1 ml aliquots in freezing medium at -20°C for 2-3 hours, then directly - with cooling to -80°C or -1°C/min if in a bubble container - Freeze at 80°C. In another embodiment, the 1.0x enzyme mix can be digested at block 102 for 16 minutes.

2 shows a flow diagram of another method 200 for dissociating tissue by isolating microvessels using concentrated or purified enzymes. Method 200 is similar to method 100 of FIG. 1 , wherein a further option at step 208 includes the use of a post digestion wash, wherein the post digestion wash occurs before the second centrifugation. Accordingly, blocks 202, 204, 206, and 210 of FIG. 2 are similar to blocks 102, 104, 106, and 110 as described above. With respect to the new block 208, a post-disintegration wash may be a feature adjacent to digestion comprising the use of 0.01% porcine gelatin. In one embodiment, washing after digestion may comprise the use of an enzyme inhibitor. Enzyme inhibitors may include, but are not limited to, classes of inhibitors such as peptide inhibitors, small molecule inhibitors, natural matrix substance inhibitors, or combinations thereof. Natural matrix substance inhibitors may include, but are not limited to, collagen gel, fibrin, elastin, gelatin, or combinations thereof.

Example 2

The method of Example 2 begins with sterile consumables and follows best biosafety level 2 (BSL-2) laboratory practices and aseptic techniques. Consumables may include:

● A 20㎛ (~φ60mm) nylon mesh filter cut round to fit the bottom of a 100mm Petri dish.

● A 500㎛ (~φ100mm) nylon mesh filter cut into a 10cm square larger than the bottom of a 100mm Petri dish.

● Wire stainless steel mesh screen

● 125 ml polycarbonate Erlenmeyer flask with one small stir bar per flask

● 100mm x 20mm Petri dish

● 50ml conical centrifuge tube

● Steri-flip filter (FISHER SCIENTIFIC Cat# SCGP00525)

● 200μl Large Orifice Micropipette Tips

● 1000μl pipette tips

● Serological pipettes - 25, 10 and 5 ml

● Benchtop Centrifuge

● Shaking type hot water pot

● 0.1% bovine serum albumin protein (BSA) as blocking buffer in cationic free phosphate buffered saline (PBS) (BSA-PBS, BSA FISHER SCIENTIFIC Cat# BP1605-100)

● Collagen-made gold (VITACYTE Cat# 011-1060)

● DNase - bovine pancreatic deoxyribonuclease 1 (Sigma DN25)

● 2% gelatin solution in PBS, autoclaved

● Frozen Medium (FISHER SCIENTIFIC Cat# 12648010)

● Hanks Balanced Salt Solution (HBSS, FISHER SCIENTIFIC Cat# 14175103)

The procedure of Example 2 with reference to FIG. 2 is as follows:

A 20 μm nylon screen is placed in a Petri dish containing 20 ml of BSA-PBS for later use. For block 202, no more than 35 ml of fat is transferred to a 50 ml conical tube. You can use up to 4 conical fat tubes at a time. The volume of each tube is 50 ml with HBSS. Invert the tube to wash, centrifuge at 400 g for 4 min to make the fat on top of the conical tube the top fat layer. Transfer the top fat layer to a new 50 ml conical tube (up to 35 ml per new tube), increase the volume to a maximum of 50 ml with HBSS, and centrifuge the fresh tube at 400 g for 4 min. Place no more than 30 mL fat per 125 mL Erlenmeyer flask containing a small spin bar. A first fat-enzyme solution is prepared through measurement and preparation of collagenase gold and DNase solutions. A first fat-enzyme solution is prepared containing 1 mg/ml of each enzyme in BSA-PBS (ie, 15 mg collagenase and 15 mg DNase in 15 ml BSA-PBS). A volume equal to the volume of fat to be decomposed and an excess of 0.5Xml are prepared. A 0.2 μm SteriFlip filter was used to sterilize the first fat-enzyme solution.

Next, 1 ml enzyme solution is added per ml fat in the flask (ie 15 ml enzyme is added to 15 ml fat washed). Tighten the cap of the flask and wrap it with parafilm. For the first digestion of block 202, transfer the flask to a 37°C water bath and shake for 1 minute, then continue incubation in the water bath without shaking for an additional 7 minutes.

For the first centrifugation operation of block 204, transfer the first fat-enzyme solution into 50 ml conical tubes (35 ml or less per tube) and bring the volume of each tube to 50 ml with BSA-PBS. Next, 250 μl of sterile 2% gelatin is added to each conical tube (for a final concentration of 0.01% gelatin) and a first centrifugation run occurs at 400 g for 4 min. After centrifugation, the pellet produces a conical tube containing blood cells, stromal cell mass and microvessels.

At block 206, the fat from the conical tube is collected at the top (where the fat will appear undissolved as the upper fat layer) and transferred to the same 50 ml Erlenmeyer flask with a stir bar. A new enzyme mixture in the same volume as the second fat-enzyme solution is added as in the first digestion (ie 15 ml if starting with 15 ml of fat). The flask is capped with Parafilm, and the flask is incubated in a 37° C. water bath for 8 minutes with shaking in the second digestion. During the breakdown of fat in the second digestion, a 50 mL conical tube can be used to aspirate the remaining liquid over each pellet, leaving about 5 mL on the pellets. The pellet can be resuspended and transferred to a Petri dish.

At block 208, the resulting fat-enzyme solution may be transferred to a 50 ml conical tube and filled to 50 ml with BSA-PBS. Also, add 250 μl of 2% gelatin to each conical tube (for a final concentration of 0.01% gelatin) and centrifuge at 400 g for 4 minutes in a second centrifugation operation. The resulting pellet contains isolated microvessels and non-degradable matrix elements. The lipid/supernatant and supernatant can be aspirated leaving about 5 ml over the pellet placed at the bottom of the tube.

At block 210, each pellet is resuspended and 25-30 ml of BSA-PBS is added. The pellet is added to a Petri dish containing microvessels (eg, pellet) generated in the first digestion. Using sterile forceps, place a metal screen on top of a new Petri dish and a 500 μm nylon mesh filter on top of the metal screen. Remove undigested tissue fragments from the suspension by slowly pipetting the microvascular suspension from the dish onto a 500 μm screen. When complete, rinse the Petri with 10 ml BSA-PBS and pipette onto a 500 μm screen. Rinse the screen by pipetting an additional 20 ml of BSA-PBS onto the screen to wash any microvessels through the screen or screen attached to any tissue mass. The 500 μm screen is discarded and the wire support is transferred to a new 10 cm dish. At this stage, microvessels pass through the screen and are present in the dish.

Using sterile forceps, place a 20 μm nylon mesh filter on top of the wire mesh screen and place it in the center of a new base dish. The filtered microvessel suspension in a Petri dish currently used for 500 μm screening is pipetted through, for example, a 20 μm screen in concentric circles. If the suspension begins to move very slowly through the screen, puddles form on the screen and it takes time to move, stop this step and proceed to the next step. This slow motion and puddles indicate a high yield of microvessels so that every single cell cannot be washed out if the step continues. Instead, retrieve a second 20 μm screen, briefly immerse in BSA-PBS, and continue the pipetting step with additional screens as needed. The original dish is rinsed with 10 ml of BSA-PBS to remove any remaining microvessels, and pipetted with a screen.

Rinse the screen again by gently pipetting 20 ml BSA-PBS concentrically, taking care not to exceed the edges of the screen to wash away any remaining single cells. At this stage, microvessels are trapped at the top of the screen with single cells passing through the screen.

Using sterile forceps, slide the 20 μm nylon mesh filter from the wire mesh and place it in a Petri dish containing 20 mL BSA-PBS that was originally used to submerge the screen, with the microvessels facing up. Soak the 20㎛ mesh for 10 minutes. Gently shake the Petri dish back and forth and remove the microvessels from the nylon mesh filter. The top of the nylon screen is cleaned by pipetting a portion of the microvessel suspension around the screen and then pipetted onto the screen. Transfer this suspension to a 50 ml tube. Pipette 10 ml of fresh BSA-PBS to the screen at a time, then transfer the rinse water to a 50 ml tube and repeat the wash several times. The edges of the screen wells should also be washed. The screen should be checked under a microscope to ensure that all microvessels have been removed, and if necessary, these removal steps should be repeated until all microvessels have been removed from the screen. The resulting isolated microvessels should be in a 50 ml conical tube at this stage.

The resulting isolated microvessels can now be counted. The counting procedure may include first determining that the top of the 50 ml conical tube is tight. Next, gently invert the tube containing the fragment suspension 2-3 times to ensure that the microvessels are evenly distributed in the solution. Two 20 μL samples of suspension are removed immediately after tube inversion and streaks are formed across the glass slide. Glass slides are not sterile, so pipette tips are changed between each sample. Also, make sure there are no large drops on the pipette tip before streaking on the slide. Under the microscope, microvessels are counted in each stripe. Microvessels that have peeled off (no more cells attached) should not be counted. Each branch of a large vessel should be counted as a separate vessel. For small capillaries, only the third capillary should be counted.

Next, the total number of microvessels can be calculated through Equation 1:

Total number of microvessels = (average number of fragments of two 20 μl samples) * [(volume of suspension in 50 ml tube)/0.02)] (Equation 1)

The fragment suspension can be centrifuged at 400 g for 4 min, which can be done while counting microvessels. The supernatant can be decanted or aspirated to leave approximately 100 µL of solution over the microvascular pellet. Microvascular fragments can be loosened by gently pipetting 100 µL of the solution.

The resulting isolated microvessels can also be frozen. Cryo-tubes can be prepared such that all tubes are marked with contents (human microvessels (MV), stromal vascular fraction (SVF), etc.), lot number, number of microvessels in the vial, and author's initials. The freezing medium is brought to room temperature and the microvessels are suspended in the freezing medium in the desired amount per ml. If freezing medium is not available, DMEM (DULBECCO MODIFIED EAGLE MEDIUM) cell culture medium containing 20% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO) can be used. Transfer up to 1 ml of microvessel freezing medium suspension to each freezing tube, ensuring that the tube is capped tightly, and the microvessels are resuspended between all cryotubes. Place the cryogenic tube in a freezing container (eg MR. FROSTY available from THERMO FISHER SCIENTIFIC) and immediately transfer to a -80°C freezer. If reefer containers are not available, foam shipping containers can be used instead. If a foam container is used, the freezing tube can be stored at -20°C for 2-4 hours and then stored in a -80°C freezer or other refrigeration device according to the instructions. After 24 hours, the vials are transferred _{to liquid N 2 for storage for integrity.}

another embodiment

The concentrated or purified enzyme methods described herein provide for the development of novel isolation, culture and preparative standards for dissociating tissues by isolating human (or other) microvessels using concentrated or purified enzymes. Other embodiments are within the scope of the present disclosure. By way of example and not limitation, in examples involving a single digestion of 8 minutes using the concentrated or purified enzymes described herein, the yield is not as high as when using double digestions. Also, in examples involving various concentrated or purified enzyme concentrations for 8 minutes, the yield is not as high as when using double digestion and a significant amount of fat remains undigested. In an embodiment utilizing two double digests at 8 min each, high yields occur after the second digest with good microvascular quality. Multiple iterations of this double digestion yield consistent yields and superior quality from a variety of fat sources, such as three different fat sources.

When using the dual degradation method as described herein in Figure 1 prior to freezing, collagen can be rapidly degraded into microvascular disruption due to carryover of residual collagenase to the microvascular isolate. However, microvessels frozen for more than 4 weeks do not result in collagen degradation. Post-solution washing of block 208 immediately prior to the second digestion of block 206 and prior to the second centrifugation operation to inhibit or inactivate residual collagenase and reduce production time for microvessel use prior to 4 weeks to maintain collagen integrity during the incubation period This produces the following results and prevents high microvascular yield and collagen degradation.

Other wash protocols are also tested in various other examples. After screening, adding a cysteine (non-synthetic inhibitor) wash causes the collagen to degrade rapidly on the first day with microvascular disruption. Testing different concentrations of the enzyme resulted in a lower yield of microvessels at lower concentrations, but still a rapid breakdown of collagen on the first day with microvessel collapse. Additional PBS washes to wash away excess enzymes after enzymatic digestion still allow for rapid breakdown of collagen on the first day with microvascular disruption. Washing with diluted collagen after screening for competitive binding to the remaining enzyme results in collagen polymerization during washing, resulting in a suspension of collagen clumps with microvessels disrupting the overall structure of the collagen plug. After screening for 5 min, a 0.01% gelatin wash slows but does not stop collagen degradation, resulting in poor quality microvascular growth. Although collagen was not degraded, 0.01% gelatin added to all screens/washes after degradation reduces yield due to gelatin-dependent agglomeration of the selected microvessels. After screening for 20 min, a 0.01% gelatin wash maintains collagen integrity during the incubation period. After enzymatic digestion, only one wash with 0.1% gelatin produces high yield, good microvascular quality and non-degradable collagen.

In embodiments using fetal bovine serum (FBS) containing media, microvascular growth may be lot dependent and may involve expensive and time consuming lot testing. The use of serum-free medium (SFM) allows for a defined, cost-effective process that is lot-independent and does not contain animal products, which is useful for human studies. Microvessels do not grow with Sato SFM and 10 ng/ml VEGF. Using modified dose calculations in the recipe for components other than Sato SFM and 10 ng/ml VEGF slows the growth of microvessels. With Sato SFM and 50 ng/ml VEGF, microvessel growth was good in some of the lots tested and microvessel growth was slow in others, which may be due to gelatin culture repeats. Using a medium based on human plasma components results in good microvascular growth similar to using Sato SFM and 50 ng/ml VEGF, but it is a time-consuming medium to make.

In the example tested, testing was performed on 10K microvessel aliquots at 60,000 microvessels/mL, the 10K microvessel aliquots being customer sized. However, since human microvessels are smaller than the tested murine microvessels, their density is low for the same number of microvessels/mL, and a high density is required for robust microvessel growth. A higher number of microvessels per aliquot allows for an easier uniform suspension and more accurate counts upon thawing, and a minimum aliquot size of 20 K microvessels can be easier to use and provide consistent handling. A minimum density of 80K microvessels/mL can be used.

In the examples tested, some batches of fat contain muscle cells (eg, myocytes) that can rupture during culture resulting in microvascular death. Percoll centrifugation can separate cells of different sizes, but it does not separate microvessels with a density similar to myocytes. Since myocytes can stick to the plastic, only the top three-quarters of the isolate can be selected and the myocytes are captured by the plastic, which significantly lowers the myocyte count, improves microvascular growth and slightly lowers the microvascular yield. In embodiments, tissue culture adhesion plastics may be utilized to further promote myocyte adhesion.

3 shows a system 300 for implementing a computer-based control scheme to automate the methods 100 and 200 of the flowcharts of FIGS. 1 and 2 . System 300 may be implemented using, for example, a graphical user interface (GUI) 202 accessible from a user workstation (eg, computing device 324 ). Computing device 324 may be a smart mobile device, which may be a smart phone, tablet, or similar portable hand-held smart device. Computing device 324 includes a processor, memory communicatively coupled to the processor, and machine-readable instructions stored in the memory. The machine readable instructions cause the system 300 to follow one or more of the blocks of the methods 100 and 200 of FIGS. 1 and 2 when executed by a processor such that one or more steps of the methods 100 and 200 may be automated. can Accordingly, the machine readable instructions, when executed by a processor, may cause the system 300 to follow one or more control schemes as described in one or more processes described herein.

The system 300 includes a communication path 302 , one or more processors 304 , a memory component 306 , a software tools component 312 , storage or database 314 , artificial intelligence component 316 , network interface hardware 318 , server 320 , network 322 , and at least one computing device 324 . The various components of system 300 and their interactions will be described in detail below.

In some embodiments, system 300 is implemented using a wide area network (WAN) or network 322 , such as an intranet or Internet, or other wired or wireless communication network, which may include a cloud computing based network configuration. The workstation computing device 324 may include digital systems and other devices that allow connection to and discovery of networks, such as the smart mobile device 200 . Other system 300 variations that allow for communication between various geographically diverse components are possible. The lines shown in FIG. 3 represent communication, not physical connections, between the various components.

As noted above, system 300 includes a communication path 302 . The communication path 302 may be formed of any medium capable of transmitting a signal, such as, for example, conductive wires, conductive traces, optical waveguides, or the like, or a combination of media capable of transmitting a signal. Communication path 302 communicatively connects the various components of system 300 . As used herein, the term “communicatively coupled” means that the connected components exchange data signals with each other, such as, for example, electrical signals through conductive media, electromagnetic signals through air, optical signals through optical waveguides, and the like. means you can

As noted above, system 300 includes processor 304 . Processor 304 may be any device capable of executing machine readable instructions. Accordingly, the processor 304 may be a controller, integrated circuit, microchip, computer, or any other computing device. The processor 304 is communicatively coupled to other components of the system 300 by a communication path 302 . Accordingly, communication path 302 may communicatively couple any number of processors to each other, and may enable modules coupled to communication path 302 to operate in a distributed computing environment. In particular, each module may act as a node capable of transmitting and/or receiving data. Processor 304 may process input signals received from system modules and/or extract information from such signals.

As noted above, system 300 includes a memory component 306 coupled to communication path 302 and communicatively coupled to processor 304 . Memory component 306 may be a non-transitory computer-readable medium or non-transitory computer-readable memory and may be configured as a non-volatile computer-readable medium. The memory component 306 may include RAM, ROM, flash memory, a hard drive, or any device capable of storing machine readable instructions such that the machine readable instructions may be accessed and executed by the processor 304 . . Machine readable instructions may include, for example, machine language that may be directly executed by a processor or assembly language, object oriented programming (OOP), scripting language that may be compiled or assembled into machine readable instructions and stored in memory component 306 . , logic or algorithm(s) written in any programming language, such as microcode. Alternatively, the machine readable instructions are written in a hardware description language (HDL), such as logic implemented via a field-programmable gate array (FPGA) configuration or an application-specific integrated circuit (ASIC) or equivalent. can be Accordingly, the methods described herein may be implemented in any conventional computer programming language, either as pre-programmed hardware elements or as a combination of hardware and software elements. In an embodiment, system 300 includes a processor communicatively coupled to memory component 306 that stores instructions that, when executed by processor 304, cause the processor to perform one or more functions as described herein. 304) may be included.

With continued reference to FIG. 3 . As noted above, system 300 includes a display, such as a GUI on the screen of computing device 324 , to provide visual output, such as, for example, information, graphical reports, messages, or combinations thereof. Computing device 324 may include one or more computing devices across platforms or may be communicatively coupled to devices across platforms, such as smart mobile device 200 including smart phones, tablets, laptops, and the like. A display on the screen of the computing device 324 is coupled to the communication path 302 and communicatively coupled to the processor 304 . Accordingly, the communication path 302 communicatively couples the display to other modules of the system 300 . A display may comprise any medium capable of transmitting light output, such as, for example, a cathode ray tube, a light emitting diode, a liquid crystal display, a plasma display, and the like. Additionally, it is noted that the display or computing device 324 may include at least one of a processor 304 and a memory component 306 . Although system 300 is shown in FIG. 3 as a single integrated system, in other embodiments the system may be a standalone system.

System 300 is provided herein with a software tool component 312 for automating steps of one or more protocols or methods as described herein and to result in more accurate, consistent and higher yields of microvascular growth and isolation. and an artificial intelligence component (316) for learning and providing the neural network with machine learning capabilities that can provide machine learning to automatically improve and modify the applied automated protocol or method as described in In one embodiment, the machine readable instructions cause the system 300, when executed by the one or more processors 304, to do at least the following: to automate one or more protocols of the system 300 as described herein. Applying artificial intelligence component 316 to train the neural network model used by system 300 , with higher yields of microvessel growth and increasing accuracy of isolation, and consistency by system 300 over time. Apply machine learning to the neural network model via artificial intelligence component 316 to modify one or more protocols over time based on historical data associated with one or more protocols in system 300 to achieve The software tools component 312 and the artificial intelligence component 316 are coupled to the communication path 302 and communicatively coupled to the processor 304 . Processor 304 may process input signals received from system modules and/or extract information from such signals.

Data stored and manipulated in system 300 as described herein is utilized by artificial intelligence component 316, which utilizes cloud computing-based network configurations such as the cloud for machine learning. and artificial intelligence can be applied. This machine learning application creates models that can be applied by system 300, making machine learning applications more efficient and intelligent when executed. By way of example and not limitation, artificial intelligence component 316 may include a component selected from the group consisting of an artificial intelligence engine, a Bayesian inference engine, and a decision engine, and may have an adaptive learning engine further comprising a deep neural network learning engine. have.

System 300 includes network interface hardware 318 for communicatively coupling system 300 with a computer network, such as network 322 . Network interface hardware 318 is coupled to communication path 302 such that communication path 302 communicatively couples network interface hardware 218 to other modules of system 300 . Network interface hardware 318 may be any device capable of sending and/or receiving data over a wireless network. Accordingly, the network interface hardware 318 may include a communication transceiver for transmitting and/or receiving data according to any wireless communication standard. For example, the network interface hardware 318 may communicate via wired and/or wireless computer networks such as, for example, Wi-Fi, WiMAX, Bluetooth, Infrared Communication (IrDA), Wireless USB, Z-Wave, Zigbee, etc. may include a chipset (eg, antenna, processor, machine readable instructions, etc.) to

With continued reference to FIG. 3 , data resulting from various applications executing on computing device 324 may be provided from computing device 324 to system 300 via network interface hardware 318 . Computing device 324 may be any device having network interface hardware 318 and hardware (eg, chipset, processor, memory, etc.) for communicatively coupling with network 322 . Specifically, computing device 324 may include an input device having an antenna for communicating over one or more of the wireless computer networks described above.

Network 322 may include any wired and/or wireless network, such as, for example, a wide area network, a metropolitan area network, the Internet, an intranet, cloud 323 , a satellite network, and the like. Accordingly, network 322 may be utilized as a wireless access point by computing device 324 to access one or more servers (eg, server 320 ). Server 320 and any additional servers, such as cloud servers, generally include a processor, memory, and a chipset for communicating resources over the network 322 . Resources may include, for example, providing processing, storage, software and information from server 320 to system 300 via network 322 . It is also noted that server 320 and any additional servers may share resources with each other via network 322, such as via, for example, a wired portion of a network, a wireless portion of a network, or a combination thereof. .

Systems and methods utilizing concentrated or purified enzymes and dual digestion features as described herein have significantly improved isolated microvascular composition, lot-to-lot consistency, and cost-effectiveness compared to previous methods using crude enzymes as described above. provides

It is noted that, for purposes of describing and defining this disclosure, reference herein to a variable or other variable that is a “function” of a parameter does not refer to only the variable or function of the enumerated parameter. Rather, reference herein to a variable that is a “function” of a listed parameter is intended to be open-ended such that the variable may be a function of a single parameter or a plurality of parameters.

It is also noted that references to “at least one” element, element, etc., in this specification should not be used to form an inference that alternative use of the articles “a” or “an” should be limited to a single element, element, etc. Pay attention.

It is noted that references herein to elements of the present disclosure to be “configured” or “programmed” in a particular way to implement particular properties or function in a particular manner are structural references as opposed to references to their intended use. . More specifically, references in this specification to the manner in which a component is “configured” or “programmed” are indicative of the component's pre-existing physical state, and therefore should be regarded as an explicit reference to the structural characteristics of the component.

Terms such as “preferably,” “generally,” and “typically,” when used herein limit the scope of the claimed disclosure or indicate that certain features are important, essential, or even critical to the structure or function of the claimed disclosure. Note that it is not used to mean Rather, such terminology is merely intended to identify particular aspects of embodiments of the present disclosure or to highlight alternative or additional features that may or may not be utilized in particular embodiments of the present disclosure.

For purposes of describing and defining this disclosure, the terms "substantially" and "approximately" are utilized herein to denote a degree of inherent uncertainty that may be attributed to any quantitative comparison, value, measurement, or other representation. Note that it will be The terms "substantially" and "approximately" are also used herein to denote the extent to which a quantitative expression may differ from a stated statement without altering the basic function of the subject matter in question.

Although the subject matter of the present disclosure has been described in detail with reference to specific embodiments thereof, the various details disclosed herein may be incorporated into various Note that it should not be construed as implying that it relates to an element that is an essential component of an embodiment. Further, it will be apparent that modifications and variations are possible without departing from the scope of the present invention, including but not limited to the embodiments defined in the appended claims. More specifically, while some aspects of the disclosure are recognized herein as preferred or particularly advantageous, it is contemplated that the disclosure is not necessarily limited to these aspects.

Note that one or more of the following claims use the term "wherein" as a transitional phrase. For the purpose of defining the present disclosure, this term is introduced in the claims as an open-ended transition used to describe a set of features of a structure and in a manner similar to the more commonly used open-ended preamble term "comprising" Note that it should be interpreted as

Claims (20)

A system for dissociating tissue by isolating microvessels using a concentrated enzyme, the system comprising:
one or more processors;
non-transitory memory communicatively coupled to the one or more processors; and
machine-readable instructions stored in the non-transitory memory that, when executed by the one or more processors, cause the system to perform at least the following operations as one or more protocols, wherein the at least the following operations:
decomposing the fat pulverized in the first digestion with a concentrated enzyme to produce a first fat-enzyme solution;
centrifuging the first fat-enzyme solution from the first digest in a first centrifugation operation to produce one or more first pellets and an upper fat layer disposed over the one or more first pellets;
decomposing the upper fat layer with the concentrated enzyme in a second digestion to produce a second fat-enzyme solution;
centrifuging the second fat-enzyme solution from the second digestion in a second centrifugation operation to produce one or more second pellets; and
passing one or more portions of the one or more first pellets and the one or more second pellets through one or more screens to create a plurality of discrete microvessels. The method of claim 1,
wherein the concentrated enzyme comprises a concentrated or purified enzyme comprising a low protease, concentrated collagenase product produced from Clostridium histolyticum for isolating cells from tissue. According to claim 1,
The machine readable instructions, when executed by the one or more processors, cause the system to at least:
and washing the second fat-enzyme solution with an enzyme inhibitor in a post digestion wash before the second centrifugation operation. 4. The method of claim 3,
wherein the enzyme inhibitor of the post digestion wash comprises one or more peptide inhibitors, one or more small molecule inhibitors, one or more natural matrix substance inhibitors, or a combination thereof. According to claim 1,
The machine readable instructions, when executed by the one or more processors, cause the system to at least:
washing said one or more parts with a gelatin solution; and
and passing the one or more portions through two screens to create the plurality of discrete microvessels. 6. The method of claim 5,
The machine readable instructions, when executed by the one or more processors, cause the system to at least:
and cryopreserving the plurality of isolated microvessels. According to claim 1,
The machine readable instructions, when executed by the one or more processors, cause the system to at least:
and using an additional enzyme as a catalyst for degradation of the first fat-enzyme solution. 8. The method of claim 7,
wherein the additional enzyme comprises deoxyribonuclease (DNase). According to claim 1,
The machine readable instructions, when executed by the one or more processors, cause the system to at least:
Suction the upper lipid layer and the supernatant of the second fat-enzyme solution from the second centrifugation operation to further perform the operation of leaving the one or more second pellets at the bottom of the tube containing the second fat-enzyme solution to do, system. According to claim 1,
The machine readable instructions, when executed by the one or more processors, cause the system to at least:
and milling the fat to produce milled fat through the use of manual mixing, a liposuction cannula, or a combination thereof. 11. The method of claim 10,
wherein the fat is received from one or more treatments comprising liposuction, abdominoplasty, or a combination thereof. According to claim 1,
The machine readable instructions, when executed by the one or more processors, cause the system to at least:
and applying an artificial intelligence component to train a neural network model used by the system to further automate the one or more protocols of the system. 13. The method of claim 12,
The machine readable instructions, when executed by the one or more processors, cause the system to at least:
Applying machine learning to the neural network model via the artificial intelligence component to modify the one or more protocols over time based on historical data related to the one or more protocols in the system, resulting in the system over time A system that further performs operations that provide higher yields of microvessel growth and isolation with increased precision and consistency. A method of dissociating tissues by isolating microvessels using a concentrated enzyme, the method comprising:
digesting the fat pulverized in the first digestion with a concentrated enzyme to produce a first fat-enzyme solution;
centrifuging the first fat-enzyme solution from the first digest in a first centrifugation operation to produce at least one first pellet and an upper fat layer disposed over the at least one first pellet;
digesting the upper fat layer with the concentrated enzyme in a second digestion to produce a second fat-enzyme solution;
centrifuging the second fat-enzyme solution from the second digestion in a second centrifugation operation to produce one or more second pellets; and
passing one or more portions of the one or more first pellets and the one or more second pellets through one or more screens to create a plurality of discrete microvessels. 15. The method of claim 14,
and washing the second fat-enzyme solution with an enzyme inhibitor in a post digestion wash before the second centrifugation operation. 16. The method of claim 15,
wherein the enzyme inhibitor of the post digestion wash comprises one or more peptide inhibitors, one or more small molecule inhibitors, one or more natural matrix substance inhibitors, or a combination thereof. 17. The method of claim 16,
wherein the enzyme inhibitor of the post digestion wash comprises 0.01% porcine gelatin. 15. The method of claim 14,
and using an additional enzyme as a catalyst for degradation of the first fat-enzyme solution. A method of dissociating tissues by isolating microvessels using a concentrated enzyme, the method comprising:
digesting the fat pulverized in the first digestion with a concentrated enzyme to produce a first fat-enzyme solution;
using an additional enzyme as a catalyst for degradation of the first fat-enzyme solution;
centrifuging the first fat-enzyme solution from the first digestion in a first centrifugation operation to produce at least one first pellet and an uppermost fat layer disposed over the at least one first pellet;
centrifuging the first fat-enzyme solution from the first digest in a first centrifugation operation to produce at least one first pellet and an upper fat layer disposed over the at least one first pellet;
digesting the upper fat layer with the concentrated enzyme in a second digestion to produce a second fat-enzyme solution;
washing the second fat-enzyme solution with an enzyme inhibitor in the post digestion wash, wherein the enzyme inhibitor of the post digestion wash comprises one or more peptide inhibitors, one or more small molecule inhibitors, one or more natural matrix substance inhibitors, or a combination thereof. Included -;
centrifuging the second fat-enzyme solution from the second digestion in a second centrifugation operation to produce one or more second pellets; and
passing one or more portions of the one or more first pellets and the one or more second pellets through one or more screens to create a plurality of discrete microvessels. 20. The method of claim 19,
wherein the enzyme inhibitor of the post digestion wash comprises 0.01% porcine gelatin.

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